A C T A U N I V E R S I T A T I S L O D Z X E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 7, 1990
Zofia Banati-Grueaka, Tadeuez Krajeweki, Pauet Nowak
AMI NO ACID C OMPO SITION AND N-TER MIN AL RESIDUES OF GOOSE, DUCK AND HEN PROTH ROMBIN
Prothrombin obtained from oxylated goose, duck and hen plasma was purified by filtration on Sephadex G-25, chromatography on hy- droxyapatite and DEAE-Sephadex A-50. Specific activities of isola-ted preparations were found to be 610-650 NIH/mg. Goose as well as duck and hen prothrombin preparations displayed alanine as N-ter- minal residue similarly to mammalians. Amino acid analysis of these proteins showed a higher content of aspartic and glutami acids in all the three kinds of prothrombin in comparison with mammalian one.
INTRODUCTION
Prothr omb in is an inactive form of thrombin (E.C.3. 4.21.5) w hi ch plays a pivotal role in the final stages of blood co ag
u-lation by conve rting fibrinogen to fibrin. In the past few years very extensive studies on mamm alian prothr ombin have been performed, Seegers [17-18] and Mag nuss on [13-14 ]. Human or bovine pro thrombin is a glycoprotein [15-16] consisting of. a single po lype ptide chain wit h mole cular weigh t of about 70 000 [l, 6, 11, 12], Alanine is the N-terminal residue for prot hro m-bin of so far investigated mam malians [man, ox, horse, sheep, dog, r a t ] and serine constitutes the C-terminal amino acid £ 4, 20 ]. Di chroism spectrum analysis indicates that 75% of the m o -lecule is of a r andomcoil structure and 5% is an oC-helix [3,
7 ]. The studies of physi cochemical and biological properties of avian prothrombin are by far less advanced. Ther efo re It ap pe-ared interesting to identify the N-ter minal residues and amino acid composition of goose, duck and hen prothrombin.
MATERIALS AND METHODS
Isolation of p rothrom bin
Prot hrombin preparations we re ob tained from goose, duck and hen blood according to Cox and Hanahan method [5], 5 1 of fresh blood were ce ntrifuged and BaSO^ was added to plasma (.10 g/ /100 ml plasma) to adsorb prothrombin. Protein was eluted from the com ple x wit h 0.06 M sodi um citrate. The supernatant ob ta in -ed after centrifugat ion was added to powder cellulose to adsorb the accompan ying proteins, especi ally factor X. The remai ning protein contam inations were pr eci pitated by ammonium sulfate. 209 g (n h4) 2S 0 4 were added to 1000 ml of the supernatant, sti r-red for one hour and centr ifuged for 30 min. at 440 g. The appropriate protein fraction co ntaining prothrombin was salted out by adding 2 g/10 ml ammonium sulfate to the supernatant. The pr ecip itated proteins were diss olve d in the min ima l volume of 0.01 M trisodium citrate ( p H 6.5) and purified chromatogra- p h ic a ll y .
Purificat ion of the pr othr ombin preparation
P urif ication of protein fraction was carried out according to Benarous, Labie and Joss o method [ 2 ] using gel filtration and followed by chromatog raphy on hydroxyapa tite and DE AE-Seph adex A-50. Crude protein was filtrated on Sephadex G-25 column (2.5 x x 100 cm) using 0.15 M pot assium phosp hate buffer at pH 6.8 for elution. In the course of separation one main peak containing prothro mbin and 2-3 peaks mos t prob ably the degra dation p r o -ducts of this protein we re obtained. Afterwards the ma in peak ,<ias purified by hydro xyapatite chromatography. The eluate from
Sephadex G-25 (lOO mg protein) was applied to a column (2.4 x x 20 cm ) with hydroxyap atite and elution was performed in a linear gradient of 0.2-0.5 M potas siu m phosph ate buffer (500 ml in each chamber) at a flow rate of 60 ml/hr. DEAE-Sepha dex A-50 chromat ography was the final stage carried out in the lineai gradient of 0.0-0.7 M NaCl in potas sium phosphate buffer (350 ml in each chamber). 20-25 mg of protein was applied to a column (2.5 x 30 cm). Pure protein was eluted with salt solution at the concentr ation 0.4-0.5 M NaCl. The ob tained fractions c on-taining prothr ombin were pooled together and thickened until fi-nal concentra tion 1 mg/ml.
Determination of the activity of thrombin preparations
The activity of thrombin preparations was determ ined by two- stage (Jackson, Thom as and Hanahan) metho d Lio]. Prothrombin wa: conv erted into thrombin by incubation (37°) with thromboplastii and calcium ions for 20 rrin. and next its activity was d e te r -mine d on the basis of clotting ability of 0.1% fibrinogen s o l u -tion.
Identification of N-terminal amino acids
Goose, duck and hen preparations of pro thrombin were subjec to DNS proc edu re L 8 , 9]. 50 nmoles of lyophilized protein we re di solved in 0.3 ml of wat er and 0.15 ml of 0.4 M ph osphate buffer and 250 mg urea. Afterwards 0.25 ml of di methylfo rmamid and 0.^5 ml oi
5-dir,iethylaminonaphtalinsulfonsaure-l-chlorid in acetone were a d-ded to the above solution. Next it was carefully stirred and
left in a dark room for 30 min. at 20°C. Then the mixture v>as diluted four times by adding 3 ml of water and 4 ml of 10% T C A . The formed sediment was centrifuged (10 min., 2000 g), washed twice wit h acetone and ether, and dried. Finally 0.25 ml of 5.5 M HC1 were aaded to the sediment. Hydrolysis was carried out in flooded tubes for 6 h at 110°C. The hydrolyzate was diluted with wate r and evapo rated until the elimination of HC1. The rest was diss olv ed in 0.1 ml of acetone: 1 M HC1 mi xture 1 : 1 v/v.
Thinlayer ch rom atography according to Sthal [19 ]
Glass plates (20 x 20) covered wi th 0.1 m m silica gel layer were actlvited for 1 b at 110°C. Hyd rolyzate and amino acid s am-ples wer e applied to each plate. Separation was carried out in two systems : 1) benzene : piridine : acetic acid (16 : 4 ¡ 1 ) , 2) toluene : 2-chloro ethanol : ammonia 28% (6 : 10 : 4). After d e -ve loping the chrom atogram in the first system the plates were dried at 110°C and fluorescence was evoked by UV to identify N-terminal amino acid of the investigated proteins.
A u t om atic amino acid an al ysis
Th e analysis of the proteins was prec eeded by acid h yd ro -lysis. 0.25 ml of 5.5 M HC1 wer e added to 1 m g protein. H yd ro ly -sis was don e in flooded glass tubes for 24, 48 and 72h at 110°C. The hydrolyz ates were evap orated until dr y under the same co n -ditions. The procedure was repeated several times to remove 1IC1. Amin o acid composition of the protein hydrolyzate was de termined on Joel automatic analyzer of amino acids.
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RESULTS AND DISCUSSION
Isolated prot hrombin preparations from goose, duck and hen plasma after filtration on Sephadex G-50 yielded always one ma in peak corresponding to the prothrombin fraction and 2-3 other small ones repr esenting protein contami nations (Fig. l).
' Chroma togr aph y of the prothro mbin fraction on hydroxy apati- te resulted in four di stinct peaks. The last one, eluted v'ith phosphate buffer at a conce ntration 0.3-0.5 M, r epr esented mo re purified, goose proth rombin fraction (Fig. 2).
Afte rwar ds the pr oth romb in peak was further separated on DE AE-S ephadex A-50 column. The fraction eluted in the range of 0.3-0.4 M NaCl contained pure and active pr oth rombin pr epa ra -tion, moving in polyac ryl ami de gel as a single band (Fig. 3, 4 a). Analogical patterns were ob tained for duck and hen p ro
-thrombins. Their electrophoret ical mobili ties are shown in Fig. 4 A and 4 B.
Fig. 1. Purification of crude goose prothrombin on Sephadex G-25 (for details see Materials and methods)
Fraction num ber
Fig. 3. Chromatography of goose prothrombin on DEAE-Sepliadex A-SO
Fig. 4. Polyacrylamide disc electrophoresis of goose (a), duck (b) and hen (<D pure prothrombin preparations
Biologi cal activity of all investigated bird prothrombins, was found to be 610-650 NIH/mg protein (Tab. l). The DNS p ro -cedure according to Cros and Laboussa was done in order to de termine the N-t erminal residues. The DNS amino acid d e ri -vatives of amino acids present in the hydrolyzate were iden-tified by thinlayer chromatography. The results indicate that alanine is the N-termi nal amino acid for all three kinds of prothr ombin (Fig. 5). Compara tive amino acid analysis of all kinds of prothrom bin preparat ions carried out by automatic analyser following acid hydrolysis is shown in Tab. 2.
T a b l e 1 Specific activity of prothrombin preparations measured by two stage text according to Jackson, Thomas and Hanahan method
Number of NIH/mg protein
preparation
hen goose duck
1 656 623 610 2 644 648 602 3 648 630 620 4 649 636 598 5 652 628 626 6 « 636 650 612 7 649 623 600 8 678 > 646 628 9 650 628 596 10 652 626 620 Average 651 .4 634.1 61 1 .2 Standard deviation 10.7 9.9 1 1 .9 • u • N
___ iS an
»SOLVENT II TAIa •Val Val Ala -I Ser SPOT OH 123 •••T a b l e 2
Amino acid composition of avian prothrombin
Amino acids
amino acids/1 mol protein
hen goose duck
Asp 59.2 61 .8 60.7 Tre 32.1 29.9 31.5 Ser 27.8 28.9 26.9 Glu 68,6 69.1 67.4 Pro 32.0 29.5 30.0 Gli 39.8 40.4 39.5 Ala 30.2 29.6 29.4 Wal }.1'.0 29.8 29.1
Met ślad ślad ślad
lie 20.4 21.2 19.8 Leu 39.8 37.9 39.0 Typ 18.9 21 .3 22.6 Fen 19.0 18.8 17.9 His 13.8 14.2 14.0 Liz 44.1 43.5 45.1 Arg 24.8 26.3 27.0 Trp 14.1 15.0 -Asp + Glu Liz+His+Arg 1.54 1.55 1.49
Trypto phan was determ ined spectro photomertica lly after alkaline h yd r ol y si s .
Summar izi ng all above results it can be stated that: 1. Prot hrombin pr epa rations of goose, duck and hen possess the bi ological ac tivity in the range of 610-650 NIH/mg proteins that is lower in comparison with the mamm alia n one (about 3000 N IH/m g protein).
2. Al anine is a common N-ter minal amino acid.
3. Prothr ombins of inves tigated birds are quite comparable in respect of amino acid compo sition as well as the ratio of acid and base amino acid (l.49 - 1.55).
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Department of Biochemistry University of Łódź, Poland
Zofia Banaś-Gruszka, Tadeuez Krajewski, Pauet Nowak
SKŁAD AMINOKWASOWY I N-KONCOWE AMINOKWASY PROTROMBINYGĘSI, KACZEK I KUR.
Ptotrombinę otrzymywano z plazmy szczawianowej gęsi, kaczek i kur metodą Cox’a i Hanahan (1970), oczyszczano metodą Benarous, Labie i Josso (1973), sto-sując filtracje na żelu Sephadex G-25, chromatografie na hydroxyapatvcie oraz chromatografię na żelu DEAE~Sephadex A-50. W jednorodnych pod względem elek-troforę tycznym białkach, wykazujących aktywność 610-650 NIH/mg protrombiny ozna-czono N-końcowe aminokwasy. Stwierdzono, źe N-końcowym aminokwasem w prepara-tach protrombiny gęsi, kaczek i kur jest alanina, podobnie jak u ssaków (czło-wiek, koń, wół", owca, pies, szczur). Analiza składu aminokwasowego wspomnia-nych białek ujawniła wysoką zawartość kwasu asparaginowego i glutaminowego we wszystkich rodzajach protrombin.
